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1.  Recombinant Rabbit Leukemia Inhibitory Factor and Rabbit Embryonic Fibroblasts Support the Derivation and Maintenance of Rabbit Embryonic Stem Cells 
Cellular Reprogramming  2012;14(4):364-376.
The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs.
PMCID: PMC3411342  PMID: 22775411
2.  The prostaglandin E2 EP1 receptor mediates pain perception and regulates blood pressure 
Journal of Clinical Investigation  2001;107(3):325-331.
The lipid mediator prostaglandin E2 (PGE2) has diverse biological activity in a variety of tissues. Four different receptor subtypes (EP1–4) mediate these wide-ranging effects. The EP-receptor subtypes differ in tissue distribution, ligand-binding affinity, and coupling to intracellular signaling pathways. To identify the physiological roles for one of these receptors, the EP1 receptor, we generated EP1-deficient (EP1–/–) mice using homologous recombination in embryonic stem cells derived from the DBA/1lacJ strain of mice. The EP1–/– mice are healthy and fertile, without any overt physical defects. However, their pain-sensitivity responses, tested in two acute prostaglandin-dependent models, were reduced by approximately 50%. This reduction in the perception of pain was virtually identical to that achieved through pharmacological inhibition of prostaglandin synthesis in wild-type mice using a cyclooxygenase inhibitor. In addition, systolic blood pressure is significantly reduced in EP1 receptor–deficient mice and accompanied by increased renin-angiotensin activity, especially in males, suggesting a role for this receptor in cardiovascular homeostasis. Thus, the EP1 receptor for PGE2 plays a direct role in mediating algesia and in regulation of blood pressure.
PMCID: PMC199184  PMID: 11160156
3.  Deficiency of the Stress Kinase P38α Results in Embryonic Lethality 
The mitogen-activated protein (MAP) kinase p38 is a key component of stress response pathways and the target of cytokine-suppressing antiinflammatory drugs (CSAIDs). A genetic approach was employed to inactivate the gene encoding one p38 isoform, p38α. Mice null for the p38α allele die during embryonic development. p38α1/− embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38α−/− ES cells lacked p38α protein and failed to activate MAP kinase–activated protein (MAPKAP) kinase 2 in response to chemical stress inducers. In contrast, p38α1/+ ES cells and primary embryonic fibroblasts responded to stress stimuli and phosphorylated p38α, and activated MAPKAP kinase 2. After in vitro differentiation, both wild-type and p38α−/− ES cells yielded cells that expressed the interleukin 1 receptor (IL-1R). p38α1/+ but not p38α−/− IL-1R–positive cells responded to IL-1 activation to produce IL-6. Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38α1/+ and p38α−/− ES cells. Therefore, these studies demonstrate that p38α is a major upstream activator of MAPKAP kinase 2 and a key component of the IL-1 signaling pathway. However, p38α does not serve an indispensable role in apoptosis.
PMCID: PMC2195860  PMID: 10704466
inflammation; cytokines; mitogen-activated protein kinase; signaling; cytokine-suppressing antiinflammatory drug
4.  Collagen-induced Arthritis Is Reduced in 5-Lipoxygenase-activating Protein-deficient Mice 
The Journal of Experimental Medicine  1997;185(6):1123-1130.
Collagen-induced arthritis in the DBA/1 mouse is an experimental model of human rheumatoid arthritis. To examine the role of leukotrienes in the pathogenesis of this disease, we have developed embryonic stem (ES) cells from this mouse strain. Here, we report that DBA/1 mice made deficient in 5-lipoxygenase-activating protein (FLAP) by gene targeting in ES cells develop and grow normally. Zymosan-stimulated leukotriene production in the peritoneal cavity of these mice is undetectable, whereas they produce substantial amounts of prostaglandins. The inflammatory response to zymosan is reduced in FLAP-deficient mice. The severity of collagen-induced arthritis in the FLAP-deficient mice was substantially reduced when compared with wild-type or heterozygous animals. This was not due to an immunosuppressive effect, because anti-collagen antibody levels were similar in wild-type and FLAP-deficient mice. These data demonstrate that leukotrienes play an essential role in both the acute and chronic inflammatory response in mice.
PMCID: PMC2196231  PMID: 9091585

Results 1-4 (4)